Project Summary Mutations in DNA are the source of heritable genetic variation in all organisms. However, non-heritable mutations in transcripts can also occur during the process of transcription. In bacteria, such errors occur approximately 10,000-fold more frequently than mutations to DNA and are therefore pervasive across the transcriptome. An error that occurs within a transcript will be translated up to 40 times; thereby amplifying the effects of a single transcription error. Because such errors can be detrimental, bacteria have evolved multiple quality control mechanisms to minimize the amount of transcription errors that do occur. For decades, information about the fidelity of transcription was restricted to in vitro transcription assays or in vivo measurements of single reporter genes. A problem with trying to measure error rates over the entire transcriptome is that errors are introduced by sequencing technologies, complicating the differentiation of true vs. artefactual errors. We have recently applied a new RNA-sequencing technology (termed CirSeq) that can distinguish between errors arising from these two sources, allowing us, for the first time, to measure errors at every transcribed site in a genome. The proposed research will investigate multiple transcription quality control mechanisms. In Aim 1, we will investigate if mutations in sites that alter the structure of the RNAP at the RNA:DNA hybrid affect transcription fidelity. This Aim will measure the effects of these mutations on both base misincorporations and slippage across the transcriptome. In Aim 2, we will implement CirSeq to determine the contributions of the GreA, GreB, and DksA fidelity proteins to the improvement of transcriptional fidelity. In Aim 3, we will investigate if the physical coupling between transcription and translation acts to improve the fidelity of transcription. Upon completion of these aims, we will have uncovered how effective the known transcription quality control mechanisms are at increasing the accuracy of transcription. Additionally, we will investigate a potential new mechanism of improving transcriptional fidelity in protein-coding genes. Ultimately, these aims will lead to a better understanding of this fundamental cellular process that, if disrupted, can have implications in cellular aging, stress survival, and persistence to antibiotic treatment.